菊花花蕾培植體利用農桿菌轉殖花色基因後之再生

The lethal rate of leaf explants in Chrysanthemum ‘Pink Flame', ‘Red windmill',‘Margenta Linker'and ‘Linker'was up to 100％ when they were cultured in medium containing hygromycin at 25 mg/L. When hygromycin in medium decreased to 20 mg/L, the leathal rate was still over 87％. As well as they were cultured on 15 mg/L hygromycin medium, the lethal rate was over 80％, except ‘Margenta Linker'explants (66％). Floral expalnts with involucre were easier browning and relapsing than those without involucre. And the whole receptacle explants regenerated more shoots than parts of receptacle explants were easier browning and relapsing than those without involucre. And the whole receptacle explants regenerated more shoots than parts of receptacle explants regenerated. In addition, flower bud with 1cm ray florets had higher survival rate. After transforming pigment gene by Agrobacterium tumefacien-mediated, flower bud explants were more survival and less relapsing, and regenerated more shoots. Each receptacle explant of chrysanthemum of ‘Red windmill'and ‘Linker'regenerated 0.76 and 0.33, respectively. The regeneration efficiency of receptacle was higher than leaf and petiole explant.菊花'Red Windmill'、'Pink Flame'、'Linker'、'Margenta Linker'葉片培殖體培養在含hygromycin之培養基時，當hygromycin濃度達25mg/L時，死亡率達到l00%;濃度為20mg/L時，致死率仍達到87%以上;濃度在l0mg/L時，除。Margenta Linke，，培殖體致死率為66%外，其他品種致死率均達到80%以上。不同花蕾培殖體型式轉殖花色基因後再生情況，發現以完整總花托再生芽體情況最佳，其次分別為包括總苞之花蕾上半部組織或總花托之上半部。但包括總苞組織，轉殖花色基因後培植體褐化率、農桿菌復發程度均為最高。以花瓣尖端著色但未超過lcm的花蕾作為培殖體，存活培殖體數量較總苞未展開或花瓣伸長大於lcm者多。菊花器官培殖體經農桿菌轉殖花色基因後，發現以花蕾培殖體存活率較高，農桿菌復發率較低，再生芽體數量較多。培殖體使用莖段或小花梗經農桿菌轉殖花色基因後，均無法再生芽體。'Red Windmill'和'Linker'轉殖花色基因後，每月花蕾培殖體再生芽體數量分別為0.76與033，高於葉片及葉柄培殖體

The Morphology of the Primary Dental Arch of Chinese Children in Shijiazhuang-City Part-I: Research concerning the size of the primary tooth crown, primary dental arch and the condition of primary occlusion

The purpose of this study was to investigate the size of the primary tooth crowns, primary dental arches, standard values and frequency distribution of primary occlusion in Chinese children. With the cooperation of a kindergarten in Shijiazhuang-city, China, the dental plasters from 55 children (36 boys and 19 girls, age range from 3-6 years old) with normal primary occlusion were collected. Because of different growth rates of the children, these children were divided into two groups, one comprising children less than 5 years old and the other of 5 years old or older. According to the method that the Japanese Society of Pediatric Dentistry reported, the data were statistically analyzed and compared to Japanese children. The results were summarized as follows: 1. Except lower primary lateral incisors, the values of the mesio-distal width of primary crowns of each tooth were significantly larger for boys than for girls in all tooth types. Also the Chinese children were inclined to show smaller mesio-distal tooth crown width than those of the Japanese children in all types of teeth. 2. Compared with the Japanese children, the Chinese children were inclined to show smaller primary dental arch length both in the maxilla and the mandible. But in the senior group, the Chinese girls were inclined to show smaller primary dental arch width than their Japanese female counterparts. On the contrary, the Chinese children tended to show larger dimensioned primary dental arch height than those of the Japanese children. 3. Though spaces between teeth (primate and growth spaces) were found in the Chinese children, the frequency was lower than that of the Japanese children. 4. The frequency of terminal plans in the Chinese children was 41.8% in the vertical type, 6.4% in the distal-step type and 51.8% in the mesial step type. 5. The primary canines occlusal relationship in the Chinese children was 63.1% in type I, 13.6% in type II and 23.7 in type III

Collaboration with International Rice Research Institute - Development of Mutation Pool for IR64 Rice Variety

水稻基因組已完全解序，未來基因註解為重要工作，而突變體為探討基因功能最佳材料。本研究與國際稻米研究所IRRI專家合作，利用疊氮化鈉進行秈稻品種IR64之誘變建立突變庫做為基因功能探討與新特性品種開發之依據。本年度繼續進行：1.誘變後代M7與M8世代之栽培管理，性狀調查與純化；2.建立突變庫農藝性狀資料庫，供後續外表型、突變基因型及基因體分析之基本資料；3.將注重快速生長株、脆稈、香氣水稻、澱粉(糯性)組成等突變體之純化及類似野生型之返祖(revertant)植株等特性進行篩選；4.建立SSR 與AFLP基因型分析系統；5.以SSR系統進行脆稈突變品系之基因定位與分子標誌開發。做為生質能源品種之開發及建立突變基因功能分析之基礎。Rice genome has been sequenced the annotation of genomic sequences will be the most important task in the future. Mutants serve as valuable materials for gene functional studies. This project was initiated to establish a mutation pool for the most important indica variety of the world, IR64, using sodium azide mutagenesis in collaboration with the scientists at IRRI to obtain mutants for rice functional gene study and new variety development. The following investigations and experiments will be conducted this year: 1. Cultivation, phenotype investigation, and purification; 2. Establish of phenotype database for future phenotype analysis; 3. Mutants with variations in fast growth rate, brittle culm, aroma, starch mutant, and domestication related traits will be focused; 4. SSR and AFLP genotyping system will be established for mutant detection and genotyping; 5. The SSR genotyping system will be applied to map gene(s) responsible for brittle culm and to develop markers for variety development in the future. The achievements of this project will provide valuable materials, a novel mutation pool of indica rice, and system for the functional genomics study in rice

Functional Genomic Study on Aromatic Rice-Mapping, Cloning and Functional Study of Aroma Related Genes in Mutant(I)

水稻是世界主要的糧食作物，香味水稻具較高商品價值及市場潛力，其研究與利用廣獲各界重視，但目前尚未有任何顯性香味基因之種原被報導或選殖成功。本研究團隊以疊氮化鈉(sodium azide)誘變TNG67 所得之SA0420 突變品系，具有濃厚芋香，且受單一顯性基因所控制，為目前已知世界所僅有之材料，突變庫中仍有數個類似顯性香味突變體，對未來香味水稻之育種與應用極具重要性。前期研究成果歸納如下：1.育成S0420/TNG67 雜交組合之385 個香與不香之重組自交系(RILs)；2.進一步將SA0420 香味基因定位於第八染色體上之q8.1、q8.2 及q8.3 三個區域；3.選殖定序SA0420 香味基因區及與TNG67 相對應之區域共約200kb，發現此區域亦接近基因定位區，多個(9/16)基因發生突變，可能與香味突變有關；4.轉殖OsGAPDH3i 基因，確實可使轉基因植株葉片呈香，證實前期蛋白質體結果與假說。本年度計畫將依據前期研究成果，繼續進行1.利用SSR 及基因選殖之成果，設計具香味專一性之SCAR 標誌，針對S0420/TNG67 雜交組合之385 個香與不香之RILs 進行標定，完成香味基因微細定位(fine mapping)、選殖基因；2.依據基因標定之結果，完成香味基因區域之選殖、序列分析與突變之比對分析，探討香味突變之原因；3.繼續進行疑為香味基因(GSA-AT，GF14c 及GA3PDH 等)之基因轉殖及轉殖株之性狀分析，以探討其與香味之關係；4.利用已建立之蛋白質體技術2D-Western 探討轉基因植株之基因與蛋白質表現及與呈香性狀之關係助於建立基礎材料及技術優勢，以。本計畫執行成果將有：分子標誌輔助育種，育出具高品質之非GM 香味水稻品系、選殖出具有商業價值之香味相關基因、瞭解水稻香味生成之基因調控機制、篩選開發新香味突變體，進而結合各領域之資訊，建立良質香米的培育與生產模式，結合學術創造產業價值。Functional genomic study on aromatic rice –1. Mapping, cloning and functional study of aroma related genes in mutant.Rice is a crop of world important, aroma is a quality trait with highercommercial value and marketing potentiality. Therefore, the aroma study has becomea very important topic in rice production. Neither dominant aroma traits have beenreported nor genes been cloned in rice. A novel aromatic mutant, SA0420, controlledby a single dominant trait was found in a sodium azide induced mutation pool ofTNG67 rice variety by our rice research team. This dominant aroma trait is veryimportant to aroma rice breeding and has attracted great interest from the ricecommunity. The final goals of this proposal are: to study the genetic mechanismcontrols aroma, clone and study the function of the aroma gene, and apply it indeveloping aroma variety.In the previous term of this study we have the following achievements:1.Developed a recombinant inbreed lines (385 F8 lines) of SA0420/TNG67. 2.Mapped the aroma genes q8.1、q8.2 and q8.3 on chromosome 8 of SA0420. 3. Cloned,sequenced, and analyzed the 100kbs (of 128kbs) of the aroma region from SA0420and TNG67, respectively. Nine of 16 genes in this region were found to be highlymutated and related with the aroma production. 4. Transgenic plants withOsGAPDH3i (the RNAi construct for GAPDH3 gene) showed a protein reductionand aroma on leaves. 5. Establish the antibody production system for the putativearoma genes (GAPDH1~8) and high sensitive 2D-Western system for transgenicaroma plant screening.In year 2008, based on these previous results, we plan to 1. Complete the finemapping of aroma genes of SA0420 by combining SSR, SCAR markers using 385RILs. 2. Clone and characterize the genes related to the aroma production. 3.Continue the functional study on the putative aroma genes (GAPDH, GSA-AT, andGF14c) through transgenic approach and the 2D-Western technique. At the end ofthis proposal we will establish: 1. a good genetic resource for breeding aroma ricevariety with clear genetic mechanism. 2. An efficient marker-assisted selection (MAS)system for breeding aroma rice variety. 3. A know-how cultivation system for thebetter control of producing high quality aroma rice, and increasing the value of riceindustry

Quickly Breeding of Aromatic Rice Variety through Marker-Assisted Backcrossing

香米是國際市場受歡迎且價格高的稻米種類之一。現今世界香米品種的香氣特性大多遺傳自Basmati、Jasmine 或Della，且遺傳特性皆為隱性，不利於香米育種的選拔。由於香氣性狀不易選拔，因此期望利用分子標誌輔助選拔(marker-assisted selection, MAS)進行回交育種，將香氣導入優良品種(系)，育成高品質並具香氣的稻米。本研究室所建立臺農67 號突變庫具有3,400 個純系突變品系，其中全株呈香的突變品系SA0420 為世界唯一單一完全顯性香氣遺傳特性。本研究室蒐集並在同一時間種植世界八種重要香米品種(系)如： Basmati370、Jasmine 85 等與本試驗所用芋頭香之香味基因供給親(aromadonor parent)香米突變品系SA0420，比較不同品種與品系之香氣，發現所有參試香米品種之香味皆與SA0420 相同，均為芋頭香。因此SA0420 品種之香味應該可以為世界各地喜愛香米的消費者所接受。因此，利用SA0420 做為香氣基因提供親進行雜交育種，較其他隱性香氣提供親更具高選拔效力。分子標誌輔助回交(marker-assistedbackcrossing, MAB)育種可以提昇香氣導入輪迴親的成功率，並縮短香米品種的育種年限。由前期香味基因定位結果與經驗累積，我們已建立完整S0420 香味相關分子摽誌系統與技術。本研究擬將SA0420 的香氣特性分別導入自臺農67 號突變品種選育而成，具金黃色胚乳的稉稻臺農76 號品種(已授權給二廠商)，及全世界栽培面積最廣的秈稻IR64 品種，期望至少育成具香氣的黃金香米及香IR64 等二個水稲新品種，以提高水稲品種的商業價值。計劃期程內獲得的香米品種將來可以申請品種權，以技轉或授權方式釋出新品種。本研究所建立的分子標誌輔助回交系統與技術平台，將可做為未來改良水稻品種各種性狀的重要參考依據。Aromatic rice is one of popular and high market price rice grain productsin international market. The fragrance of most aromatic rice varieties in theworld was inherited from rice varieties such as Basmati, Jasmine or Della andis recessive inheritance. Due to difficult selection the recessive aroma trait isdisadvantageous for aromatic improvement in rice. Owing to the selection ofthe fragrance is not easy to manipulate, marker-assisted selection (MAS) isapplied to introduce the fragrance into elite variety (line) to breed high qualityand aromatic grain in backcrossing program. We have generated a TNG67mutant pool with 3400 pure lines, particularly aromatic mutant SA0420 iswhole-plant fragrant and has unique single complete dominant fragrantinheritance in the world. Comparing the fragrances of eight world famousaroma varieties including Basmati 370、Jasmine 85 in the same conditionsuggested that they were all smelled like taro as of SA0420. Therefore, mutantSA0420 can be a good fragrant gene donor and is better than other recessivedonors and has high selection effect in breeding program. According toprevious efforts in aroma gene mapping we have accumulated and establishedenough genomic information and experiences for breeding aroma rice.Marker-assisted backcrossing (MAB) can increase the fragrant introgressionof recurrent and decrease the time of aromatic rice breeding. In this study thefragrance of SA0420 will be introgressed into japonica type mutant varietyTNG76 with golden endosperm and indica type variety IR64 with broadcultivation area in the world. It is expected to breed at least 2 new varieties,golden-aroma rice and aromatic IR64, to increase their commercial value.Aromatic variety bred in this study will apply for plant variety rights and thenrelease through technology transfer or authorization. A model marker-assistedbackcrossing system and plateform will be established to improve allcharacteristics of rice variety

生物技術在作物品種改良上之應用

目前地球有限的耕地面積及農業生產體系，已無法負擔急速增加的世界人口。如何應用生物技術改良作物品種，提高農業生產量或產值，已成為農業研究的首要目標之一。傳統育種方法屬於雜交育種，品種改良主要受種原變異之限制，而不同物種（species）間之雜交頗為困難，育種成果難有大突破，「綠色革命」(green revolution)很難再發生。利用生物技術進行作物品種改良，係指以遺傳工程(genetic engineering)技術，將特定基因或性狀導入缺乏此基因或特性之目標作物（target crop）的育種方法；因此利用生物技術進行作物品種改良，可以突破種原之限制及種間雜交之瓶頸，創造新性狀或新品種，亦即未來「基因革命」(gene revolution）很可能迅速取代「綠色革命」。今後利用生物技術進行作物品種改良，可朝下列重點努力：創造高附加價值之轉基因作物品種；育成具環保特性之抗病、抗蟲及抗殺草劑等作物品種，減少農藥之施用；育成具耐旱、耐寒、耐熱及耐重金屬等具環境忍受性之作物品種

Development of Plant Cell Lysis and DNA Extraction System

基因組DNA 萃取為分子檢測、DNA指紋分析、親源鑑定、GMO檢測、基因表現分析及分子標誌應用等技術之主要步驟，為最耗時、費力，也最為困難之步驟，更是目前自動化分析中的瓶頸技術。本產學合作計畫擬與廠商合作開發適合植物細胞破碎之細胞破碎機，配合DNA萃取方法，組合成「植物細胞DNA萃取系統」。此一系統研發成功後，將可應用至植物、動物以及微生物等，所有生物細胞之粉碎與DNA萃取，如能與合作廠商之現有自動化系統整合，將可成為最具有競爭力之自動化植物分子標誌技術系統。Genomic DNA extraction is the major step for molecular detection, DNA fingerprinting, GMO detection, gene expression analysis, and molecular marker technique. It is also the most labor and time consuming process and becomes the bottleneck for the automation of all the plant molecular analytic system. In this project a cell lysis system will be developed for the genomic DNA extraction from plant cells. This system can be applied to the extraction of genomic DNA in plants, animals, and microbes. Integration of this system to the automatic DNA purification system will make it a most powerful DNA extraction system for the automation of plant molecular technology

Breeding for Transgenic Rice Containing Porcine Lactoferrin

水稻是本省重要的農作物之一, 其栽培面積十分廣大.但因栽培成本不斷提高, 使得種植水稻之經濟效益相對降低.加上目前政府正積極運作加入世界貿易組織, 唯市場開放後, 水稻的經濟價值即將面臨重大的入關壓力.因此, 除了研究降低生產成本之外, 提高水稻生產之附加價值, 利用水稻當作生物反應器來生產高經濟價值蛋白質或人類用藥, 成為另一條提升水稻價值之有效途徑.本計畫擬利用基因工程技術將豬乳鐵蛋白基因轉殖入水稻中, 生產高經濟價值之豬乳鐵蛋白進而提高水稻之附加價值.執行本計畫後, 預期育成含有豬乳鐵蛋白之轉基因水稻, 以開創稻米產業之新方向.本計畫若能成功, 其預期之經濟效益將相當大, 不僅可降低加入WTO後稻米市場開放之衝擊, 確保台灣水稻之永續經營, 並可使水資源有效再利用, 影響國計民生甚鉅.Rice ( Oryza sativa L. )is one of the most important and most widely cultivated crops in Taiwan.However, because the cost of cultivation is increasing every year, and the upcoming strong competition of imported rice from other countries after joining the WTO, the economic value of growing rice is downsizing.In addition to reduce the production cost, using the rice plants as bioreactors to produce pharmaceutical proteins becomes a new way for increasing economic values of rice and may solve the problems we are facing.The general purpose of this proposal is to transfer porcine lactoferrin gene into rice using genetic engineering techniques and intend to produce this recombinant protein in transgenic rice plants to increase the additional value of rice.After performing this project, we expect to obtain transgenic rice plants containing porcine lactoferrin gene and these transgenic rice plants will serve as bioreactors to produce lactoferrin to increase the economic value of rice and thus open up a new direction of our rice production in the future

Collaboration with International Rice Research Intitute - Development of Mutation Pool for IR64 Rice Variety

本計畫擬以疊氮化鈉(sodium azide)進行秈稻品種IR64種子之誘變處理，誘變後代將依據IRRI出版的水稻調查性狀手冊進行性狀調查，調查突變發生率及突變性狀之分離，並依譜系法(pedigree method)予以純化。將注重快速生長株(high biomass)、巨大株(giant plant)、脆稈(brittle culm)、可能是C4型及類似野生型之返祖(revertant)植株等性狀，進行觀察與相關生化與分子層次分析系統之建立。預計調查IR64之M1植株至少3,000株。調查完的資料以EXCELL軟體進行分析，分析其突變率及各性狀突變情形，建立品系及性狀矩陣，建立譜系資料(pedigree)，供後續外表型、突變基因型及基因體分析之基本資料及查詢。In this project we will collaborate with IRRI and aim to establish a mutation pool for the indica rice variety IR64 using sodium azide as a mutagen. The phenotype of rice mutants will be investigated according to the Rice trait book of IRRI and establish a data base using Excell software for further analysis. Pedigree method will be applied to keep tracing the phenotype during generations until the traits are fixed. High biomass, giant plant, brittle culm, and possible trait for C4 type plant will be focused in our investigation and will be applied to molecular and biochemical analysis. At least 4000 plants will be investigated in the M1 generation to obtain the basic variation information of this mutation pool for further study